U.S. patent number 4,816,559 [Application Number 07/071,264] was granted by the patent office on 1989-03-28 for biologically active peptides tan-866.
This patent grant is currently assigned to Takeda Chemical Industries, Ltd.. Invention is credited to Setsuo Harada, Nozomi Katayama, Hideo Ono.
United States Patent |
4,816,559 |
Harada , et al. |
March 28, 1989 |
Biologically active peptides TAN-866
Abstract
The iron-containing biologically active peptide TAN-866 produced
by microorganisms belonging to the genus Pseudomonas and its iron
free compounds have antibacterial activity mainly against
gram-negative bacteria. These peptides can be used as a therepeutic
agent for bacterial infections in mammals, domestic fowl, etc.,
caused by Pseudomonas aeruginosa. Further, TAN-866 and its deacyl
compounds are also promising as the starting materials and
intermediates for the synthesis of novel products.
Inventors: |
Harada; Setsuo (Kawanishi,
JP), Ono; Hideo (Kobe, JP), Katayama;
Nozomi (Kobe, JP) |
Assignee: |
Takeda Chemical Industries,
Ltd. (Osaka, JP)
|
Family
ID: |
26423115 |
Appl.
No.: |
07/071,264 |
Filed: |
July 8, 1987 |
Foreign Application Priority Data
|
|
|
|
|
Jul 8, 1986 [JP] |
|
|
61-160439 |
Apr 1, 1987 [JP] |
|
|
62-82095 |
|
Current U.S.
Class: |
530/317;
435/71.3; 930/190; 930/270; 530/333; 930/200 |
Current CPC
Class: |
A61P
31/04 (20180101); C12N 1/205 (20210501); C07K
7/06 (20130101); C12R 2001/385 (20210501); Y10S
930/19 (20130101); Y10S 930/27 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
7/00 (20060101); C07K 7/06 (20060101); A61K
38/00 (20060101); C07K 007/50 (); C07K 001/12 ();
C12P 021/00 (); C12P 021/04 () |
Field of
Search: |
;530/317,333
;435/68,71 |
Other References
Haskell et al., The Journal of Antibiotics, vol. XVI, No. 2, 1963,
pp. 67-75..
|
Primary Examiner: Phillips; Delbert R.
Assistant Examiner: Mohamed; Abdel A.
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Claims
What we claim is:
1. A compound of the formula, or an iron-free compound thereof:
##STR4## wherein R.sub.1 is H or OH and each R.sub.2, R.sub.3 and
R.sub.4 is H or CH.sub.3.
2. The compound according to claim 1, wherein each R.sub.1,
R.sub.2, R.sub.3 and R.sub.4 is H.
3. The compound according to claim 1, wherein R.sub.1 is OH and
each R.sub.2, R.sub.3 and R.sub.4 is H.
4. The compound according to claim 1, wherein R.sub.1 is H, any two
of R.sub.2, R.sub.3 and R.sub.4 are H and the other is CH.sub.3,
and the retention time of HPLC is 5.8 minutes.
5. The compound according to claim 1, wherein R.sub.1 is H, any two
of R.sub.2, R.sub.3 adn R.sub.4 are H and the other is CH.sub.3,
and the retention time of HPLC is 6.2 minutes.
Description
The present invention relates to novel peptides TAN-866 A, B, C or
D or their related compounds useful as a therapeutic agent for
bacterial infectious diseases, a method of producing them and a
microorganism capable of producing at least one species of TAN-866
A, B, C and D.
As the compound most resembling to TAN-866 A, B, C and D in
physico-chemical properties as described later can be mentioned
succinimycin [Journal of Antibiotics, Vol. 16, p. 67 (1963)].
Owing to the development of therapeutics using antibiotics,
diseases caused by bacteria have been overcome for the most part.
There are, however, still some serious problems to be solved in the
field of therapeutics of infectious diseases. For example,
long-term or high-dose medication with conventional antibiotics
causes changes in the flora of disease-causative bacteria
(replacement of bacteria) and advent of drug-resistant bacteria
(aquisition of drug-resistance) or increase of opportunistic
microorganisms due to lowering of autoimmunity, resulting in an
increase in diseases. In order to solve these problems, such
substances as possessing novel structures and showing novel
biological activities or intermediates for synthesizing them have
always been demanded.
The present inventors isolated a great number of microorganisms
from soils and plants for the purpose of searching for new
substances, and investigated the substances produced by those
microorganisms, finding that microbes of certain species produce a
novel substance, that the microbes belong to the genus Pseudomonas,
and that these microbes are capable of accumulating in a culture
medium a substance possessing antibacterial activity against
principally gram-negative bacteria. The present inventors isolated
these substances, and, on the basis of their physicochemical
properties as well as biological properties, they confirmed that
these substances were novel and decided to name them TAN-866 A, B,
C and D, respectively.
Based on these findings, the present inventors made further studies
to complete the present invention.
Namely, the present invention relates to:
(1) A compound of the formula [I], or an iron-free compound
thereof: ##STR1## wherein R.sub.1 is H or OH and each R.sub.2,
R.sub.3 and R.sub.4 is H or CH.sub.3,
(2) A method for producing a compound of the formula [I], or an
iron-free compound thereof which comprises cultivating on a culture
medium a microorganism belonging to the genus Pseudomonas and
capable of producing at least one species of compounds represented
by said formula to allow at least one species of said compounds to
be accumulated in the medium, recovering thus-accumulated product,
followed by subjecting the product to iron-liberation, upon
necessary,
(3) Pseudomonas fluorescens capable of producing a compound of the
formula [I],
(4) A compound of the formula [II], or an iron -free compound
thereof: ##STR2## wherein R.sub.1 is H or OH and each R.sub.2,
R.sub.3 and R.sub.4 is H or CH.sub.3, and
(5) A method of producing a compound of the formula [II] or an
iron-free compound thereof, which comprises subjecting a compound
of the formula [I] to hydrolysis by alkaline solutions for the
cleavage of the lactone bond and to hydrolysis by the amidases for
elimination of CH.sub.3 (CH.sub.2).sub.5 --CH.dbd.CH--CH.sub.2
--CO-- group, followed by subjecting the product to
iron-liberation, upon necessary.
In the present specification, the compounds represented by the
formula [I] are sometimes referred to briefly as TAN-866 A, B, C or
D correspoonding to meanings of R.sub.1, R.sub.2, R.sub.3 and
R.sub.4 as described below:
TAN-866 A: each R.sub.1, R.sub.2, R.sub.3 and R.sub.4 is H
TAN-866 B: R.sub.1 is OH, and each R.sub.2, R.sub.3 and R.sub.4 is
H
TAN-866 C: R.sub.1 is H, any two of R.sub.2, R.sub.3 and R.sub.4
are H and the other is CH.sub.3, and the retention time of HPLC
mentioned later is 5.8 minutes.
TAN-866 D: R.sub.1 is H, any two of R.sub.2, R.sub.3 and R.sub.4
are H and the other is CH.sub.3, and the retention time of HPLC
mentioned later is 6.2 minutes.
TAN-866 A, B, C and D are sometimes generally called "TAN-866", and
the compounds which are obtained by iron-liberation from the
corresponding compounds of the formula [I] are sometimes referred
to as "iron-free compounds". Further, the compounds represented by
the formula [II] are sometimes referred to as "deacyl-TAN-866 A, B,
C and D" and their corresponding "iron-free compounds".
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1, 2, 3 and 4 are, respectively, infra red absorption
spectrum of TAN-866 A, B, C and D.
FIGS. 5, 6, 7, and 9 are respectively, IH NMR spectrum of TAN-866
A, B, C and D in D.sub.2 O.
As the TAN-866 producing microbes employable in the present
invention, any ones belonging to the genus Pseudomonas and capable
of producing TAN-866 can be mentioned, for example, Pseudomonas
fluorescens. More completely, Pseudomonas fluorescens YK-310 strain
isolated from soils collected at Zentsuji, Kagawa Prefecture, Japan
(hereinafter sometimes abbreviated as "strain YK-310").
Bacteriological characteristics of strain YK-310 are as
follows:
(a) Morphology
Morphological characteristics were observed after incubation on a
meat-extract agar slant medium at 24.degree. C. for 5 days.
Cell shape and size: Rod, 0.6.about.1.2 .mu.m diameter
0.8.about.2.1 .mu.m length Motile with polar multitrichous
flagellation; No sporulation; Gram-negative.
(b) Growth on various media
Observation was conducted for 1 to 14 days under incubation at
24.degree. C.
1 Nutrient agar plate:
Colonies are colorless, opaque and circular. The colony surface is
head-like. The colony margin is sinuous. No diffusible pigment is
produced.
2 Nutrient agar slant:
Abundant, glossy and unfolded-cloth like, opaque and colorless.
3 Nutrient broth:
Grows in turbid suspension. Forms a thin pellicle. No precipitation
appears.
4 Gelatin stab:
Good growth mainly on the upper portion. Liquefaction is
observed.
5 Litmus milk:
Litmus-reduction activity is not observed. Peptonization activity
is observed but coagulation is not.
(c) Physiological characteristics
1 Nitrate reduction:-
2 Denitrification:-
3 MR (methyl red) test:-
4 VP (Voges-Proskauer) test:-
5 Indole production:-
6 Hydrogen sulfide production (TSI agar and lead acetate
paper):-
7 Starch hydrolysis:-
8 Citrate utilization (Koser's Christensen's and Simon's
medium):+
9 Inorganic nitrogen source utilization:
(i) Potassium nitrate:+
(ii) Ammonium sulfate:+
10 Pigment production (King's A, King's B and Mannitol yeast
extract agar medium): Production of yellowish green diffusible
pigment is observed in King's B medium. No production of diffusible
pigment is observed in either King's A medium or yeast extract agar
medium.
11 Urease:+
12 Oxidase:+
13 Catalase:+
14 Conditions for growing:
(i) pH: 4.7.about.10.0, optimally 7.2.about.8.4
Medium: glucose 0.1%, yeast extract 0.01%, ammonium sulfate 0.1%,
sodium chloride 0.1%, magnesium sulfate (7 hydrate) 0.05%,
phosphate buffer 0.1M (sterilized separately)
(ii) Temperature: 10.degree..about.34.degree. C., optimally
10.degree..about.30.degree. C. Medium: bouillon liquid medium
15 Oxygen demand: aerobic
16 O-F (oxidative-fermentative) test [Hugh.Leifson method]:
oxidative
17 Acid and gas production from sugars and their utilization:
______________________________________ Acid Gas (Peptone
Utilization (Peptone Water) Water) (Davis' Medium)
______________________________________ L-arabinose + - + D-xylose +
- .+-. D-glucose + - + D-mannose + - + D-fructose - - + D-galactose
+ - + Maltose - - .+-. Sucrose - - + Lactose - - .+-. Trehalose - -
- D-sorbitol - - .+-. D-mannitol - - + Inositol - - + Glycerol - -
+ Starch - - + ______________________________________
18 G+C (guanine-cytosine) content of DNA: 65.9%.+-.1.0% (Tm
method)
19 Decomposition of polysaccharide:
carboxymethyl cellulose:-
colloidal chitin:-
sodium arginate:-
20 Decomposition of Tween 80:+
Strain YK-310 having the afore-mentioned bacteriological
characteristics was collated with bacterial species described in
Bergey's Manual of Determinative Bacteriology, 8th edition,
International Journal of Systematic Bacteriology, Vol. 30, pp.
225.about.420 (1980) and ibid Vol. 32, pp. 146.about.149; this
strain was assumed as belonging to the genus Pseudomonas, based on
the following characteristics, i.e. the strain is an gram-negative
rod, motile with multitrichous flagellation, aerobic,
catalase-positive and oxidase-positive, and the G+C content of its
DNA is 65.9.+-.1.0 mole %.
According to said Bergey's Mannual of Determinative Bacteriology,
the genus Pseudomonas is divided into four sections, i.e. Sections
I, II, III and IV, by its characteristics concerning requirement of
growth factors, intracellular accumulation of
poly-.beta.-hydroxybutyrate, utilization of DL-arginine and growth
at 40.degree. C.
Table 1 shows the characteristics of the strain YK-310 as obtained
by further experiments.
TABLE 1 ______________________________________ Characteristics of
Strain YK-310 Tests Result* ______________________________________
Poly-.beta.-hydroxybutyrate accumulation - Arginine dihydrase +
Pigment production: -King's A medium - King's B medium +
Denitrificaition - Gelatin hydrolysis + Poly-.beta.-hydroxybutyrate
hydrolysis - Utilization of carbon sources**: Sucrose + L-Arabinose
+ Propionate - Butyrate - Propylene glycol - Ethanol -
______________________________________ *+: Positive, -: Negative
**Stainer's medium [described in Journal of General Microbiology,
Vol. 43 pp. 159.about.271 (1966)] was used.
It was considered appropriate that the strain YK-310 belongs to the
Section I on the basis of the facts that the strain has no
auxotrophy and does not accumulate poly-.beta.-hydroxybutyrate
intracellularly.
Ten species are included in Section I. As the strain YK-310
produces fluorescent pigment and possesses arginine dihydrolase,
the strain YK-310 was considered as belonging to any of Pseudomonas
aeruginosa, Pseudomonas putida, Pseudomonas fluorescens,
Pseudomonas chlororaphis and Pseudomonas aureofaciens.
The strain YK-310 was different from Pseudomonas aeruginosa and
Pseudomonas chlororaphis in denitrification. It was also different
from Pseudomonas putida in hydrolysis of gelatin and utilization of
sucrose, and from Pseudomonas aureofaciens in producibility of
non-fluorescent pigment and reduction of nitrate. The
characteristics of the strain YK-310 were in good agreement with
those of Pseudomonas fluorescens. Therefore, the strain YK-310 was
identified as Pseudomonas fluorescens, and designated Pseudomonas
fluroescens YK-310.
The above-mentioned Pseudomonas fluorescens YK-310 has been
deposited at Fermentation Research Institute, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry (FRI, 1-3, Higashi-1 Chome, Yatabe-cho, Tsukuba-gun,
Ibaraki Prefecture, Japan) under the accession number of FERM
P-8833 as from July 3, 1986, and also at the Institute for
Fermentation, Osaka (IFO, 2-17-85, Juso-honmachi, Yodogawa-ku,
Osaka, Japan) under the accession number of IFO 14516 since June
24, 1986. The above deposition at FRI has been converted to a
deposit under the Budapest Treaty under the accession number of
FERM BP-1369.
Bacteria belonging to the genus Pseudomonas used in method of the
present invention are, in general, very susceptible to mutagens,
e.g., it can be varied easily by mutations using ultraviolet ray,
X-ray, chemicals (e.g. nitrosoguanidine and ethyl
methanesulfonate), etc.; and strains which can be used in the
present invention include all mutants capable of producing
TAN-866.
In the incubation of TAN-866-producing bacteria, substances which
can be assimilated by the bacteria are used properly as carbon
sources: glucose, fructose, galactose, soluble.starch, dextrin,
oils and fats (e.g. soybean oil, olive oil, etc.), organic acids
(e.g. citric acid, succinic acid, gluconic acid, etc.), etc. As
nitrogen sources, organic nitrogen compounds such as soybean flour,
cotton seed powder, corn.gluten.meal, dried yeast, yeast extract,
meat extract, peptone, urea, etc. Inorganic salts such as sodium
chloride, potassium chloride, calcium carbonate, magnesium sulfate,
potassium primary phosphate and potassium secondary phosphate,
which are essential to ordinary bacterial cultures, can be properly
used singly or in combination.
Heavy metals such as ferrous sulfate and copper sulfate, and
vitamins such as vitamin B.sub.1 and biotin, are supplemented when
required. Antifoaming agents such as silicone oil and polyalkylene
glycol ether, and surface active agents, can also be added to the
medium. Further, any other organic or inorganic substance which
facilitate the growth of microbes and thus promote TAN-866
production can also be added upon necessity.
As for culture methods, ordinary production methods for antibiotics
can be applied; either solid or liquid culture may be applicable.
In the case of liquid cultures, stationary cultures, agitating
cultures, shaking cultures, aeration cultures, etc. can be
optionally conducted; agitating culture under aeration is
especially preferable. Culture temperature is preferably in a range
of about 15.degree. C..about.32.degree. C., pH is in a range of
about 5.about.8, and the culture is conducted for approximately
8.about.168 hours, preferably 24.about.144 hours.
For harvesting the objective TAN-866 from cultures, separation
methods which are usually used to isolate metabolites produced by
microbes from their cultures can properly be used. For example,
TAN-866, which is a neutral substance, is contained mainly in
culture filtrate, and it is recovered advantageously by, among
others, the following procedures. Namely, the whole culture broth,
after addition of a filter aid, is subjected to filtration or
centrifugation to remove cells, and the resulting culture filtrate
is put in contact with a water-immiscible organic solvent to
extract the active components, or the culture liquid is put in
contact with a proper carrier to adsorb active components in the
filtrate and recover the objective products by desorbing with an
appropriate solvent fractionally by means of chromatography. The
carrier to be employed advantageously includes silica gel,
cellulose, adsorptive resins, etc. which utilize the difference of
adsorbability among compounds, or molecular sieve carriers which
utilize the difference of molecular weight among compounds.
Elutions which can be used in a proper combination to elute
objective compounds from these carriers include organic solvents,
water-containing solutions of water-soluble organic solvents, e.g.
hydrous acetone, hydrous alcohols, etc., though the combination
varies with types and properties of carriers. Depending on cases,
crude products thus obtained chromatographycally are subjected to
reversed-phase HPLC for separation to perform further
purification.
To describe in more detail, use is made of, as the carrier, for
example, Amberlite XAD-II (Rohm & Haas Co., USA), Diaion HP-10,
HP-20 and SP-207 (Mitsubishi Chemical Industries, Ltd., Japan) etc.
to adsorb the active substances in the filtrate, then the
thus-adsorbed materials are eluted with a mixture of an organic
solvent and an aqueous solution, i.e. a mixture of acetone or
methanol or the like and water or an aqueous or buffer solution
containing acids or salts.
TAN-866 can also be extracted from its aqueous solution with an
organic solvent which can be separated from water, e.g. n-butanol,
iso-butanol, n-amyl alcohol, iso-amyl alcohol, etc. Further,
TAN-866 can be adsorbed on a carrier such as silica gel or
molecular sieve type carrier e.g. Kieselgel 60 (E. Merck AG, W.
Germany) or molecular sieve carriers such as Sephadex LH-20
(Pharmacia Fine Chemicals, Sweden) and then thus-adsorbed material
can be eluted with a suitable organic solvent, for example,
chloroform, ethyl acetate, acetone, alcohols (e.g. methanol, etc.)
or a mixture thereof.
As the column to be used for reversed phase HPLC, use is made of,
for example, YMC gel (Yamamura Chemical Laboratories, Japan). As
the mobile phase, use is made of a mixture of methanol or
acetonitrile, etc. and a buffer solution. For purification of
TAN-866, besides combinations of the above-mentioned procedures, an
optional combination of concentration, crystallization,
lyophilization, etc. which are conventionally used in laboratories
can be applied.
TAN-866 presents in the culture broth as a trivalent iron complex,
and it can be purified and isolated as it is by means of procedures
described as above. Thus-isolated iron complex can be converted to
TAN-866 iron-free compound by using a conventional iron ion
removing agent such as 8-hydroxyquinoline, or a strong
cation-excahnge resin such as Amberlite IR-120 (Rohm & Haas
Co., USA), Dowex 50W (Dow Chemical Co., USA), etc. Addition of a
trivalent iron compound, e.g. ferric chloride or ferric sulfate or
the like to an aqueous solution of the free compound of TAN-866
affords TAN-866.
Physical and chemical properties of TAN-866 A, B, C and D, which
were obtained in Example 1 and 2 to be shown later are as
follows:
TAN-866A
(1) Appearance: Reddish orange solid
(2) Specific rotation: [.alpha.].sub.D.sup.25 +170.degree. (c=0.1,
in water)
(3) Molecular formula: C.sub.51 H.sub.82 N.sub.13 O.sub.19 Fe
(4) Elemental analysis (%): Samples were subjected to analysis
after drying on phosphorus pentoxide at 40.degree. C. for 6 hours.
(calculated as containing 5 moles of water)
______________________________________ C H N O Fe Found: 45.86 6.67
13.68 5.0 Calcd.: 46.15 6.99 13.72 28.93 4.2l
______________________________________
(5) Molecular weight: m/z 1237(M+H).sup.+ (SI-MS method)
(6) Ultraviolet and visible (UV & VS) absorption spectrum (in
water): .lambda..sub.max 423.+-.3 nm (E.sub.1.sup.1% .sub.cm
=25.+-.5)
(7) Infrared (IR) absorption spectrum: in KBr Main absorptions are
as follows. (FIG. 1) 3350, 2950, 1750, 1660, 1530, 1460, 1380,
1240, 1040, 980, 720, 550 (cm.sup.-1)
(8) Composition of constituent amino acids:
(a) Samples hydrolized in 6N HCl at 110.degree. C. for 15 hours:
serine (2 moles), glycine (3 moles), alanine (1 mole). valine (1
mole)
(b) Samples hydrolized in 57% hydriodic acid at 100.degree. C. for
15 hours: serine (2 moles), glycine (3 moles), alanine (1 mole),
valine (1 mole), ornithine (3 moles)
(9) HPLC:
Column: YMC-PAK A312 (Yamamura Chemical Laboratories)
Mobile phase: 36% CH.sub.3 CN water,
Flow rate: 2 ml/min. Rt=5.3 (min.)
(10) Solubility:
Soluble: water, dimethyl sulfoxide, methanol
Sparingly soluble: n-hexane, diethyl ether
(11) Classification of substance: neutral substance
TAN-866B
(1) Appearance: Reddish orange solid
(2) Specific rotation: [.alpha.].sub.D.sup.25 +164.degree. (c=0.1,
in water)
(3) Molecular formula: C.sub.51 H.sub.82 N.sub.13 O.sub.20 Fe
(4) Elemental analysis (%): Samples were subjected to analysis
after drying on phosphorus pentoxide at 40.degree. C. for 6 hours.
(calculated as containing 6 moles of water)
______________________________________ C H N O Fe
______________________________________ Found: 45.07 6.88 13.47 3.0
Calcd.: 45.00 6.96 13.38 30.56 4.10
______________________________________
(5) Molecular weight: m/z 1253(M+H).sup.+ (SI-MS method)
(6) Ultraviolet and visible (UV & VS) absorption spectrum (in
water): .lambda..sub.max 422.+-.3 nm (E.sub.1 cm.sup.1%
=20.+-.5)
(7) Infrared (IR) absorption spectrum: in KBr Main absorptions are
as follows. (FIG. 2) 3370, 2930, 1750, 1660, 1530, 1470, 1380,
1230, 1040, 980, 730, 560 (cm.sup.-1)
(8) Composition of constituent amino acids:
(a) Samples hydrolyzed in 6N HCl at 110.degree. C. for 15 hours:
serine (3 moles), glycine (3 moles), valine (1 mole)
(b) Samples hydrolized in 57% hydriodic acid at 100.degree. C. for
15 hours: serine (3 moles), glycine (3 moles), valine (1 mole),
ornithine (3 moles)
(9) HPLC:
Column: YMC-PAK A312 (Yamamura Chemical Laboratories)
Mobile phase: 36% CH.sub.3 CN Water
Flow rate: 2 ml/min. Rt=4.7 (min.)
(10) Solubility:
Soluble: water, dimethyl sulfoxide, methanol
Sparingly soluble: n-hexane, diethyl ether
(11) Classification of substance: neutral substance
TAN-866C
(1) Appearance: Reddish orange solid
(2) Specific rotation: [.alpha.].sub.D.sup.25 +187.degree. (c=0.1,
in water)
(3) Molecular formula: C.sub.52 H.sub.84 N.sub.13 O.sub.19 Fe
(4) Elemental analysis (%): Samples were subjected to analysis
after drying on phosphorus pentoxide at 40.degree. C. for 6 hours.
(calculated as containing 4 moles of water)
______________________________________ C H N O Fe
______________________________________ Found: 47.29 7.12 13.88 4.3
Calcd.: 47.20 7.01 13.76 27.81 4.22
______________________________________
(5) Molecular weight: m/z 1251(M+H).sup.+ (SI-MS method)
(6) Ultraviolet and visible (UV & VS) absorption spectrum (in
water): .lambda..sub.max 423.+-.3 nm (E.sub.1 cm.sup.1%
=24.+-.5)
(7) Infrared (IR) absorption spectrum: in KBr Main absorptions are
as follows. (FIG. 3) 3400, 2930, 1750, 1660, 1540, 1470, 1380,
1240, 1020, 980, 730, 560 (cm.sup.-1)
(8) Composition of constituent amino acids:
(a) Samples hydrolyzed in 6N HCl at 110.degree. C. for 15 hours:
serine (2 moles), glycin (3 moles), alanine (1 mole), valine (1
mole)
(b) Samples hydrolized in 57% hydriodic acid at 100.degree. C. for
15 hours: serine (2 moles), glycin (3 moles), alanine (1 mole),
valine (1 mole), ornithine (3 moles)
(9) HPLC:
Column: YMC-PAK A312 (Yamamura Chemical Laboratories)
Mobile phase: 36% CH.sub.3 CN water
Flow rate: 2 ml/min. Rt=5.8 (min.)
(10) Solubility:
Soluble: water, dimethyl sulfoxide, methanol
Sparingly soluble: n-hexane, diethyl ether
(11) Classification of substance: neutral substance
TAN-866D
(1) Appearance: Reddish orange solid
(2) Specific rotation: [.alpha.].sub.D.sup.25 +176.degree. (c=0.1,
in water)
(3) Molecular formula: C.sub.52 H.sub.84 N.sub.13 O.sub.19 Fe
(4) Elemental analysis (%): Samples were subjected to analysis
after drying on phosphorus pentoxide at 40.degree. C. for 6 hours.
(calculated as containing 4 moles of water)
______________________________________ C H N O Fe
______________________________________ Found: 47.10 7.07 13.80 4.2
Calcd.: 47.20 7.01 13.76 27.81 4.22
______________________________________
(5) Molecular weight: m/z 1251(M+H).sup.+ (SI-MS method)
(6) Ultraviolet and visible (UV & VS) absorption spectrum (in
water): .lambda..sub.max 423.+-.3 nm (E.sub.1 cm.sup.1% =22
.+-.5)
(7) Infrared (IR) absorption spectrum: in KBr Main absorptions are
as follows. (FIG. 4) 3400, 2940, 1750, 1660, 1540, 1370, 1240,
1010, 980, 730, 560 (cm.sup.-1)
(8) Composition of constituent amino acids:
(a) Samples hydrolized in 6N HCl at 110.degree. C. for 15 hours:
serine (2 moles), glycin (3 moles), alanine (1 mole), valine (1
mole)
(b) Samples hydrolized in 57% hydriodic acid at 100.degree. C. for
15 hours: serine (2 moles), glycin (3 moles) alanine (1 mole),
valine (1 mole), ornithine (3 moles)
(9) HPLC:
Column: YMC-PAK A312 (Yamamura Chemical Laboratories)
Mobile phase: 36% CH.sub.3 CN water
Flow rate: 2 ml/min. Rt=6.2(min.)
(10) Solubility:
Soluble: water, dimethyl sulfoxide, methanol
Sparingly soluble: n-hexane, diethyl ether
(11) Classification of substance: neutral substance Further, the
.sup.1 H NMR spectra of TAN-866 A, B, C and D in D.sub.2 O are
shown in FIGS. 5, 6, 7 and 8 respectively (400 MHz, .delta.ppm,
JEOL GX-400).
As described above, each TAN-866 has three moles of ornithine among
its constitute amino acids. From these results and their .sup.1 H
NMR spectral data, it is estimated that TAN-866 A and B have three
N.sup.5 -acetyl-N.sup.5 -hydroxy-ornithine and TAN-866 C and D have
two N.sup.5 -acetyl-N.sup.5 -hydroxy-ornithine and a N.sup.5
-propionyl-N.sup.5 -hydroxy-ornithine. It is known that the
N-hydroxyl group of said amino acids changes to N-hydroxyanion
##STR3## in the presence of trivalent iron ion and such iron ion is
liganded by three N-hydroxyanion (J. Antibiotics, 24, 830,
1974).
When TAN-866 A is stirred or allowed to stand in a basic aqueous
solution at 20.degree. to 60.degree. C., preferably 25.degree. to
50.degree. C., for 30 minutes to 8 hours, preferably 1 to 4 hours,
the lactone bond of TAN-866 A molecule is hydrolyzed to give its
carboxylic acid form compound (C.sub.51 H.sub.83 N.sub.13 O.sub.20
FeNa). The thus hydrolyzed compound can be isolated and purified as
its monosodium salt using a chromatography on Diaion HP-20 etc.
Deacyl TAN-866 A (C.sub.41 H.sub.68 N.sub.13 O.sub.19 Fe) is
obtained by hydrolysis of the above obtained carboxylic acid form
compound with an amidase which is contained in the bacterial cells
of Pseudomonas acidovorans IFO 13582. This hydrolysis is conducted
in a phosphate buffer of pH 3 to 9, preferably pH 5 to 8, at
25.degree. to 45.degree. C., preferably 30.degree. to 40.degree. C.
for 5 to 30 hours, preferably 10 to 25 hours. The amidase is used
in an amount of 5 to 15 times, preferably 8 to 12 times the weight
of the substrate. When the said enzyme is used for the reaction,
the bacterial cells are supplied either as they are or in the form,
previously treated with acetone etc.
Physical and chemical properties of deacyl TAN-866 A, which was
obtained in Example 5, are as follows:
(1) Appearance: Reddish orange solid
(2) Molecular weight: m/z 1103 (M+H).sup.+ (SI-MS method)
(3) Elemental analysis (%) (calculated as containing 7 moles of
water)
______________________________________ C H N O Fe
______________________________________ Found: 40.19 6.37 14.34 4.0
Calcd.: 40.07 6.72 14.82 33.85 4.54
______________________________________
(4) Molecular formula: C.sub.41 H.sub.68 N.sub.13 O.sub.19 Fe
(5) Visible absorption spectrum (in methanol): .lambda.max 422.+-.3
nm (E.sub.1 cm.sup.1% =26.+-.5)
(6) Infrared absorption spectrum: in KBr Main absorptions are as
follows. 3380, 3070, 2950, 1660, 1590, 1540, 1470 1380, 1240, 1050,
980, 790, 730, 560 (cm.sup.-1)
(7) Solubility: Soluble: water, dimethylsulfoxide, methanol
Sparingly soluble: ethylacetate, chloroform
(8) Classification of substance: amphoteric substance
TAN-866 B, C and D give the corresponding deacyl TAN-866 B, C and D
by a method similar to that for production of deacyl TAN-866 A. The
data of HPLC and SI-MS of those compounds are as follows:
TABLE 2 ______________________________________ Deacyl-TAN-866 A B C
D ______________________________________ HPLC* (rt) 3.3 min. 3.2
min. 5.2 min. 5.0 min. (retention time) SI-MS 1103 1119 1117 1117
(M + H).sup.+ ______________________________________ *Column: ODS,
YMCPack A312 Mobil phase: 8% acetonitrile/0.01 M phosphate buffer
(pH 6.3) Flow rate: 2 ml/min Detection: 214 nm
Composition of constituent amino acids of these deacyl compounds
are all identical with those of the corresponding TAN-866 A, B, C
and D.
According to the above data and the method described below, the
chemical formulae of TAN-866 and deacy TAN-866 were determined as
shown in the formulae [I] and [II] respectively. Namely, it is
determined based on the subtracted data by the SI-MS method from
TAN-866 to the corresponding deacyl TAN-866 and .sup.1 H NMR
spectrum [COSY method (.sup.1 H-.sup.1 H)] of iron-free TAN-866
that the fatty acid portion of TAN-866 is represented by the
formula of CH.sub.3 (CH.sub.2).sub.5 CH.dbd.CHCH.sub.2 CO--. The
sequence of the amino acids containing C- and N-terminal amino
acids was determined by subjecting each deacyl TAN-866 to an amino
acid sequencer. The binding position of the lactone group was
determined by the NOESY Method in the .sup.1 H NMR spectrum of
TAN-866 as shown in the formula [I].
The biological characteristics of TAN-866 and its iron-free
compounds are described as follows. The antibacterial activities of
TAN-866 are as shown in Table 3.
TABLE 3 ______________________________________ Minimal Inhibitory
Concentration (.mu.g/ml) (Note 1) TAN-866 Test Organism A B C D
______________________________________ Staphylococcus aureus FDA
>100 >100 >100 >100 209P Micrococcus luteus IFO 12708
>100 >100 >100 >100 Bacillus subtilis NIHJ PCI 219
>100 >100 >100 >100 Bacillus cereus FDA 5 >100
>100 >100 >100 Escherichia coli NIHJ JC 2 3.13 6.25 12.5
3.13 Salmonella typhimurium IFO 0.39 0.39 0.39 0.39 12529
Citrobacter freundii IFO 12681 6.25 12.5 12.5 25 Klebsiella
pneumaniae IFO 0.78 0.78 12.5 3.13 3317 Serratia marcescens IFO
12648 >100 >100 >100 >100 Proteus mirabilis ATCC 21100
>100 >100 >100 >100 Proteus vulgaris IFO 3988 >100
>100 >100 >100 Proteus morganii IFO 3168 >100 >100
>100 >100 Pseudomonas aeruginosa IFO 3.13 3.13 12.5 3.13 3080
Alcaligenes faecalis IFO 13111 >100 >100 >100 >100
Acinetobacter calcoaceticus IFO >100 > 100 >100 >100
13006 ______________________________________ (Note 1) Medium
composition Bacto Antibiotic Medium 3 (Difco Laboratories, USA):
17.5 g Bacto yeast extract (Difco Laboratories, USA): 5.0 g Bacto
agar (Difco Laboratories, USA): 20 g Distilled water (pH
unadjusted): 1000 ml Inoculum size: a loopful of approx. 10.sup.6
CFU/ml
Table 4 shows the therapeutic effects of TAN-866 and its iron-free
compounds to experimental infectious diseases in mice using
Pseudomonas aeruginosa P-9, by subcutaneous administration.
TABLE 4 ______________________________________ Compound ED.sub.50
(mg/kg)* ______________________________________ TAN-866A 0.57
TAN-866B 0.593 TAN-866C 0.590 TAN-866D 0.197 TAN-866A iron-free
0.44 compound ______________________________________ *Total of
three dosages
No acute toxicity of TAN-866A in mice was observed by
intraperitoneal or oral administration in a dose of 1000 mg/kg.
As clearly shown in these data, TAN-866 and its iron-free compounds
have antibacterial activity, mainly against gram-negative bacteria
while showing no toxicity in mammals for instance. Therefore,
TAN-866 or its iron-free compounds can be used in the therapeutics
of bacterial infections in humans and domestic animals (e.g. cows,
horses, pigs, etc.), domestic fowls (e.g. chickens, etc.), etc.
For using TAN-866 or its iron-free compounds as therapeutic drugs
of, for example, infectious diseases by Pseudomonas aeruginosa,
they are administered as, for example, injections dissolved in
physiological saline parenterally, subcutaneously or
intramuscularly at a dose of 0.1.about.20 mg/kg/day, preferably
0.5.about.10 mg/kg/day. And, TAN-866 or its iron-free compounds are
prepared into capsules by mixing with lactose and administered at a
dose of 0.5.about.100 mg/kg/day, preferably 2.about.50 mg/kg/day in
terms of TAN-866 or an iron-free compound thereof.
Deacyl TAN-866 has an amino group and a carboxylic group as the
derivatizable functions, which can easily be afforded N-acyl
derivatives by reacting with acid halides such as fatty acid
halides of carbon number 1 to 20 (e.g. myristyl chloride,
linoleinyl chloride or capryl chloride) in week base solution. Thus
obtainable N-acylated compounds are lactonized by the condensation
reagents, for example, DCC in proper solvents (e.g. DMF) or by
acidifying the reaction solutions. The new compounds thus obtained
may be assumed to appear antimicrobial activities against
Pseudomonas aeruginosa, for example. Thus, TAN-866 and deacyl
TAN-866 are also promising as the starting materials and
intemediates for the synthesis of novel medicinal products.
The following examples will describe the present invention in more
detail, but are not intended to limit the invention thereto. Unless
otherwise specified, % means weight/volume %.
EXAMPLE 1
Five hundred ml of a medium prepared by adding 0.5% precipitating
calcium carbonate to an aqueous solution (pH 7.0) containing 2%
glucose, 3% soluble starch, 1% raw soybean flour, 0.3% corn-steep
liquor, 0.5% Polypepton (Daigo Nutritive Chemicals, Ltd.) and 0.3%
sodium chloride in a 2 l Sakaguchi flask was inoculated with
Pseudomonas fluorescens YK-310 (FERM BP-1369; IFO 14516) grown on
an nutrient agar slant, which was subjected to reciprocal shaking
culture at 24.degree. C. for 48 hours. With the entire quantity of
the resulting culture broth was inoculated 120 l of a medium
prepared by adding 0.05% Actocol (Takeda Chemical Industries,
Ltd.), an antifoaming agent, to the above-mentioned medium in a 200
l tank. Cultivation was carried out at 24.degree. C. under aeration
of 120 l/min. and agitation at 180 rpm for 48 hours. With 50 l of
the resulting culture broth was inoculated 1200 l of a medium
prepared by adding 0.05% Actocol to an aqueous solution (pH 6.5)
containing 2% glycerol, 0.5% glucose, 0.5% Polypepton, 0.5% meat
extract (Wako Pure Chemical Industries, Ltd.), 0.1% sodium chloride
and 0.1% yeast extract (Daigo Nutritive Chemicals, Ltd.) in a 2000
l of tank. Cultivation was carried out at 17.degree. C. under
aeration of 1200 l/min. and agitation at 150 rpm for 42 hours.
The culture broth thus obtained was subjected to filtration by the
aid of Hyflo Super-Cel (Johns Manville Sales Corp.). The filtrate
(1300 l) was subjected to a column chromatography on Diaion HP-20
(50 l). The active substance was eluted with a 80% methanolic water
(350 l). The eluate was concentrated, from which methanol was
distilled off. The aqueous portion (30 l) was adjusted to pH 7,
followed by extraction with isobutanol (20 l). The extract was
washed with a 2% sodium bicarbonate solution, 0.05N hydrochloric
acid, followed by concentration of the isobutanol layer. The
concentrate (3 l) was added to n-hexane (10 l) to give
precipitates. The supernatant was separated by decantation. To the
remaining precipitates was further added n-hexane (3 l) to obtain a
crude substance (21.1 g) containing TAN-866A. The crude substance
(20.5 g) was dissolved in 50% methanolic water, and the solution
was subjected to a column chromatography on Diaion HP-20 (50-100
mesh, 1 l). The column was washed with 60% methanolic water (6 l),
then the antibiotic substance was eluted fraction-wise. The
fractions were combined and concentrated. The concentrate was
dissolved in a small volume of methanol, which was added to ether
to give powdery product (532 mg). The powdery product (1 g)
obtained by a similar procedure was subjected to a column
chromatography on Sephadex LH-20 (1 l), eluting with methanol. The
eluate was concentrated and the concentrate (530 mg) was dissolved
in water (10 ml), which was purified by means of a chromatography
on Diaion HP-20 (50-100 mesh, 200 ml). Thus obtained purified
powder (274 mg) was assumed to be a mixture of closely analogous
compounds by the peak pattern of HPLC. The powdery product (250 mg)
was then subjected to reversed-phase HPLC for separation (column:
YMC-PAK SH343; mobile phase: 30% acetonitrile/water) to collect the
peak portion of the principal component, which was concentrated to
yield reddish orange powder (64 mg) of TAN-866A.
EXAMPLE 2
On a scale similar to that in Example 1, cultivation, filtration,
HP-20 column chromatography and isobutanol extraction were carried
out, followed by subjecting the concentrate (3 l) of the extract
solution to chromatography on silica gel (1.5 l). The column was
washed with isobutanol (4.5 l), isopropanol (4.5 l) and
isopropanol:methanol (1:1) (4.5 l), followed by elution with
methanol (4.5 l). The eluate was concentrated to dryness to give a
powdery product (10.6 g). It was quantitatively determined by means
of HPLC that the powdery product contained TAN-866A (3.5 g).
Then, 31 g of the powdery product obtained by similar procedure to
the above was dissolved in 50% methanolic water (1 l), and the
solution was subjected to a column chromatography on Diaion HP-20
(100.about.200 mesh, 1 l). The columnm was washed with 50%
methanolic water (3 l) and 60% methanolic water (1 l), followed by
fractionately eluting the antibiotic substance with 70% methanolic
water (3 l) and 75% methanolic water (2 l). Each fraction was
concentrated and lyophilized, yielding Powder I (content of A: 67%,
7.6 g), Powder II (content of TAN-844 A: 48%, 8.0 g) and Powder III
(content of TAN-866 A: 34%, 4.4 g).
Then, the Powder I (7.5 g) was subjected to preparative
reserved-phase HPLC. From the column was eluted the antibiotic
substance with a solvent system of a 32% aqueous solution of
acetonitrile using YMC-PAK R-355 (25/44) (Yamamura Chemical
Laboratories). Each fraction was subjected to HPLC for analysis to
determine the amount of each component quantitatively, followed by
concentration and lyophilization to obtain the powder (1.7 g)
containing TAN-866 A and B, the powder (2.2 g) containing solely
TAN-866 A and the powder (0.33 g) containing TAN-866 A, C and D.
The powder containing TAN-866 A and B was again subjected to
preparative HPLC (column: YMC-PAK S-363 I-15, the solvent system:
the same as in the above-mentioned HPLC for separation) to thereby
isolate TAN-866 A (1.15 g) and TAN-866 B (209 mg) as reddish orange
powder. The powder containing TAN-866 A, C and D was similarly
processed to obtain TAN-866 C (70 mg) and TAN-866 D (150 mg) as
reddish orange powder.
EXAMPLE 3
In water (2 ml) was dissolved the purified powder (40 mg) of
TAN-866A obtained in Example 1, to which was added a solution of
8-hydroxyquinoline (40 mg) in methanol (2 ml). The reaction
solution was allowed to stand at 4.degree. C. for 15 hours. The
resulting black precipitates were filtered off with a filter paper.
Methanol in the filtrate was distilled off, followed by addition of
water (20 ml), which was washed with chloroform (10 ml) four times.
The aqueous portion was concentrated and lyophilized to obtain an
iron-free compound (29 mg) of TAN-866A as white powder.
Molecular formula: C.sub.51 H.sub.85 N.sub.13 O.sub.19
Elemental analysis (%): (samples dried over phosphorus pentoxide at
60.degree. C. for 8 hours, calculated as containing 3 mol. of
water.
______________________________________ Found Calcd.
______________________________________ C, 49.33 49.47 H, 7.45 7.41
N, 14.78 14.70 O, 28.42 ______________________________________
Molecular weight determined: by SI-MS method m/z 1184
(M+H).sup.+
UV spectrum: (in water) End absorption
IR spectrum: (in KBr) main absorptions are shown 3300, 2930, 1640,
1520, 1230, 570 (cm.sup.-)
EXAMPLE 4
By a procedure similar to that in Example 3, starting from purified
powdery products of TAN-866 B, C, D (20 mg, 10 mg, 20 mg,
respectively) obtained in Example 2, iron free compounds of TAN-866
B, C and D were obtained as white powdery products (20 mg, 8 mg and
19 mg, respectively). TAN-866 B iron-free compound:
Molecular formula: C.sub.51 H.sub.85 N.sub.13 O.sub.20
Elemental analysis (%): (samples dried over phosphorus pentoxide at
60.degree. C. for 8 hours, calculated as containing 2.5 mol. of
water)
______________________________________ Found Calcd.
______________________________________ C, 49.03 49.19 H, 7.24 7.28
N, 14.59 14.62 ______________________________________
Molecular weight determined: by SI-MS method m/z
1200(M+H).sup.+
UV spectrum: (in water) End adsorption
IR spectrum: (in KBr) main absorptions are shown 3330, 2940, 1665,
1530, 1240, 590 (cm.sup.-)
TAN-866 C iron free compound:
Molecular formula: C.sub.52 H.sub.87 N.sub.13 O.sub.19
Elemental Analysis (%): (samples dried over phosphorus pentoxide at
60.degree. C. for 8 hours, calculated as containing 3.5 mol. of
water)
______________________________________ Found Calcd.
______________________________________ C, 49.36 49.51 H, 7.21 7.35
N, 14.26 14.38 ______________________________________
Molecular weight determined: by SI-MS method m/z 1198
(M+H).sup.+
UV spectrum: (in water) End adsorption
IR spectrum: (in KBr) main absorptions are shown 3300, 2940, 1665,
1530, 1235, 590 (cm.sup.-)
TAN-866 D iron-free compound:
Molecular formula: C.sub.52 H.sub.87 N.sub.13 O.sub.19
Elemental analysis (%): (samples dried over phosphorus pentoxide at
60.degree. C. for 8 hours, calculated as containing 3 mol. of
water)
______________________________________ Found Calcd.
______________________________________ C, 49.71 49.87 H, 7.22 7.48
N, 14.41 14.54 ______________________________________
Molecular weight determined: by SI-MS method m/z 1198
(M+H).sup.+
UV spectrum: (in water) End absorption
IR spectrum: (in KBr) main absorptions are shown in 3400, 2940,
1665, 1530, 1240, 590 (cm.sup.-)
EXAMPLE 5
In 0.05M phosphate buffer (pH9, 150 ml) was dissolved TAN-866 A
(150 mg), and agitated at 40.degree. C. for 2 hours. The reaction
solution was subjected to a column chromatography on Diaion HP-20
(20 ml), and the elution was conducted with 50% methanolic water.
The eluate was concentrated to dryness to yield a sodium salt of
carboxylic acid form compound of TAN-866 A. SI-MS: m/z 1277
(M+H).sup.+, Molecular formula: C.sub.51 H.sub.83 N.sub.13 O.sub.20
FeNa
The thus obtained carboxylic acid form compound (150 mg) was added
to 0.05M phosphate buffer (pH 7, 150 ml) containing crude amidase
(1.5 g) which was yielded by a Pseudomonas acidovorans IFO 13582,
and then stirred at 37.degree. C. for 18 hours. The reaction
solution was centrifuged, and the obtained supernatant was adjusted
to pH 2.5, followed by extraction with ethyl acetate. This extract
solution contained the fatty acid. The aqueous layer was subjected
to a column chromatography on Diaion HP-20 (50-100 mesh, 30 ml).
The column was fractionately eluted with 5-20% methanolic water.
The fractions containing a peptide were concentrated to dryness to
yield deacyl TAN-866 A as reddish orange powder (97 mg).
EXAMPLE 6
TAN-866 B (10 mg), TAN-866 C (2 mg) and TAN-866 D (10 mg) was
hydrolyzed by a method similar to that in Example 5 to yield deacyl
TAN-866 B (5 mg), deacyl TAN-866 C (1.5 mg) and deacyl TAN-866 D
(5.7 mg), respectively.
* * * * *